Conventional and novel protein kinase C (PKC) isozymes are activated by binding of tumor promoters to their C1 domains (C1A, C1B). Recent investigations revealed that novel PKC isozymes (δ, ε, η) are involved in tumor promotion in vivo and their C1B domains play dominant roles on their activation by tumor promoters. Design of new agents with high selectivity for novel PKC C1B domains is indispensable to elucidate the precise mechanism of tumor promotion. 1-Hexyl-indolactam-V (1) was selected as a lead compound for such agents since 1 showed a binding preference for the C1B domains of novel PKC isozymes. Compound 1 existed as two stable conformers due to the cis-trans isomerization of the lactam amide function. Conversion of the indole ring of 1 to the indoline ring achieved the conformational fixation of 1. Highly improved selectivity for novel PKC C1B domains was observed in the trans-amide analogue (3), while the binding selectivity of the cis-amide analogue (2) was almost similar to that of 1. The docking simulation revealed that the binding mode of 3 with the PKCδ C1B domain is quite different from those of 1 and 2. Detailed analysis of the CH/π interaction using a PKCδ C1B mutant, in which Pro-11 was substituted for 4,4-difluoro-Pro, indicated that the CH/π interaction between the hydrogen atom at position 4 of Pro-11 and the benzene ring of 3 is critical for the binding of 3 to the PKCδ C1B domain. Based on these results, the conformationally analogous compound of 3 containing an indole ring (4), which would form effectively the CH/π interaction with Pro-11 of the PKCδ C1B domain, was designed. Compound 4 showed significant binding affinity and selectivity for the C1B domains of all novel PKC isozymes, especially for PKCδ and θ. Since PKCδ has a tumor suppresser role, 4 might be an antitumor agent as well as an analytical agent of tumor promotion.
